Fusion proteins for prodrug activation

ABSTRACT

The invention relates to compounds which contain an antigen binding region which is bound to at least one enzyme which is able to metabolize a compound (prodrug) which has little or no cytotoxicity to a cytotoxic compound (drug), where the antigen binding region is composed of a single polypeptide chain. It is advantageous for covalently bonded carbohydrates to be present on the polypeptide chain.

[0001] The invention relates to compounds which contain an antigenbinding region which is bound to at least one enzyme which is able tometabolize a compound (prodrug) which has little or no cytotoxicity to acytotoxic compound (drug), where the antigen binding region is composedof a single polypeptide chain. It is advantageous for covalently bondedcarbohydrates to be present on the polypeptide chain.

[0002] The combination of prodrug and antibody-enzyme conjugates for useas therapeutic composition has already been described in the specialistliterature. This entails antibodies which are directed against aparticular tissue and to which a prodrug-cleaving enzyme is bound beinginjected into an organism, and subsequently a prodrug compound which canbe activated by the enzyme being administered. The action of theantibody-enzyme conjugate bound to the target tissue is intended toconvert the prodrug compound into a compound which exerts a cytotoxiceffect on the bound tissue. However, studies on antibody-enzymeconjugates have shown that these chemical conjugates have unfavorablepharmacokinetics so that there is only inadequate site-specifictumor-selective cleavage of the prodrug. Some authors have attempted toremedy this evident deficiency by additional injection of an anti-enzymeantibody which is intended to bring about rapid elimination of theanti-body-enzyme conjugate from the plasma (Sharma et al., Brit. J.Cancer, 61, 659, 1990). Another problem of antibody-enzyme conjugates isthe limited possibility of preparing large amounts reproducibly andhomogeneously.

[0003] The object of the present invention was now to find fusionproteins which can be prepared on an industrial scale and are suitable,by reason of their pharmacokinetic and pharmacodynamic properties, fortherapeutic uses.

[0004] It has been found in this connection that compounds which containan antigen binding region which is composed of a single polypeptidechain have unexpected advantages for the preparation and use of fusionproteins, to which carbohydrates are advantageously attached, in prodrugactivation.

[0005] The invention therefore relates to compounds which contain anantigen binding region which is bound to at least one enzyme, where theantigen binding region is composed of a single polypeptide chain, andcarbohydrates are advantageously attached to the fusion protein.

[0006] An antigen binding region means for the purpose of the inventiona region which contains at least two variable domains of an antibody,preferably one variable domain of a heavy antibody chain and onevariable domain of a light antibody chain (sFv fragment). The antigenbinding region can, however, also have a bi- or multivalent structure,i.e. two or more binding regions, as described, for example, in EP-A-0404 097. However, a human or humanized sFv fragment is particularlypreferred, especially a humanized sFv fragment.

[0007] The antigen binding region preferably binds to a tumor-associatedantigen (TAA), with the following TAAs being particularly preferred:

[0008] neural cell adhesion molecule (N-CAM),

[0009] polymorphic epithelial mucin (PEM),

[0010] epidermal growth factor receptor (EGF-R),

[0011] Thomsen Friedenreich antigen β (TFβ),

[0012] gastrointestinal tract carcinoma antigen (GICA),

[0013] ganglioside GD_(3 (GD3)),

[0014] ganglioside GD₂ (GD₂),

[0015] Sialyl-Le^(a), Sialyl-Le^(x),

[0016] TAG72,

[0017] the 24-25 kDa glycoprotein defined by MAb L6,

[0018] CA 125 and, especially,

[0019] carcinoembryonic antigen (CEA).

[0020] Preferred enzymes are those enzymes which are able to metabolizea compound of little or no cytotoxicity to a cytotoxic compound.Examples are β-lactamase, pyroglutamate aminopeptidase, galactosidase orD-aminopeptidase as described, for example, in EP-A2-0 382 411 orEP-A2-0 392 745, an oxidase such as, for example, ethanol oxidase,galactose oxidase, D-amino-acid oxidase or α-glyceryl-phosphate oxidaseas described, for example, in WO 91/00108, peroxidase as disclosed, forexample, in EP-A2-0 361 908, a phosphatase as described, for example, inEP-A1-0 302 473, a hydroxynitrilelyase or glucosidase as disclosed, forexample, in WO 91/11201, a carboxypeptidase such as, for example,carboxypeptidase G2 (WO 88/07378), an amidase such as, for example,penicillin 5-amidase (Kerr, D. E. et al. Cancer Immunol. Immunther.1990, 31) and a protease, esterase or glycosidase such as the alreadymentioned galactosidase, glucosidase or a glucuronidase as described,for example, in WO 91/08770.

[0021] A β-glucuronidase is preferred, preferably from Kobayasianipponica or Secale cereale, and more preferably from E. coli or a humanβ-glucuronidase. The substrates for the individual enzymes are alsoindicated in the said patents and are intended also to form part of thedisclosure content of the present application. Preferred substrates ofβglucuronidase are N-(D-glycopyranosyl)benzyloxycarbonylanthracyclinesand, in particular, N-(4-hydroxy3-nitrobenzyloxycarbonyl)doxorubicin anddaunorubicin β-D-glucuronide (J. C. Florent et al. (1992) int.Carbohydr. Symp. Paris, A262, 297 or S. Andrianomenjanahary et al.(1992) Int. Carbohydr. Symp. Paris, A 264, 299).

[0022] The invention further relates to nucleic acids which code for thecompounds according to the invention. Particularly preferred is anucleic acid, as well as its variants and mutants, which codes for ahumanized sFv fragment against CEA (carcinoembryonic antigen) linked toa human β-glucuronidase, preferably with the sequence indicated in Table1 (sFv-huβ-Gluc).

[0023] The compounds according to the invention are prepared in generalby methods of genetic manipulation which are generally known to theskilled worker, it being possible for the antigen binding region to belinked to one or more enzymes either directly or via a. linker,preferably a peptide linker. The peptide linker which can be used is,for example, a hinge region of an antibody or a hinge-like amino-acidsequence. In this case, the enzyme is preferably linked with the Nterminus to the antigen binding region directly or via a peptide linker.The enzyme or enzymes can, however, also be linked to the antigenbinding region chemically as described, for example, in WO 91/00108.

[0024] The nucleic acid coding for the amino-acid sequence of thecompounds according to the invention is generally cloned in anexpression vector, introduced into prokaryotic or eukaryotic host cellssuch as, for example, BHK, CHO, COS, HeLa, insect, tobacco plant, yeastor E.coli cells and expressed. The compound prepared in this way cansubsequently be isolated and used as diagnostic aid or therapeuticagent. Another generally known method for the preparation of thecompound according to the invention is the expression of the nucleicacids which code therefor in transgenic mammals with the exception ofhumans, preferably in a transgenic goat.

[0025] BHK cells transfected with the nucleic acids according to theinvention express a fusion protein (sFv-huβ-Gluc) which not only wasspecific for CEA but also had full β-glucuronidase activity (see Example5).

[0026] This fusion protein was purified by anti-idiotype affinitychromatography in accordance with the method described in EP 0 501 215A2 (Example M). The fusion protein purified in this way gives amolecular weight of 100 kDA in the SDS PAGE under reducing conditions,while molecules of 100 and 200 kDa respectively appear undernon-reducing conditions.

[0027] Gel chromatography under native conditions (TSK-3000 gelchromatography) showed one protein peak (Example 6, Fig. I) whichcorrelates with the activity peak in the specificity enzyme activitytest (EP 0 501 215 A2). The position of the peak by comparison withstandard molecular weight markers indicates a molecular weight of ≈200kDa. This finding, together with the data from the SDS PAGE, suggeststhat the functional enzymatically active sFv-huβ-Gluc fusion protein isin the form of a “bivalent molecule”, i.e. with 2 binding regions and 2enzyme molecules. Experiments not described here indicate that thefusion protein may, under certain cultivation conditions, be in the formof a tetramer with 4 binding regions and 4 enzyme molecules. After thesFv-huβ-Gluc fusion protein had been purified and undergone functionalcharacterization in vitro, the pharmacokinetics and the tumorlocalization of the fusion protein were determined in nude mice providedwith human gastric carcinomas. The amounts of functionally active fusionprotein were determined in the organs and in the tumor at various timesafter appropriate workup of the organs (Example 7) and by immunologicaldetermination (triple determinant test, Example 8). The results of arepresentative experiment are compiled in Table 4.

[0028] Astonishingly, a tumor/plasma ratio of 5/1 is reached after only48 hours. At later times, this ratio becomes even more favorable andreaches values >200/1 (day 5). The reason for this favorablepharmacokinetic behavior of the sFv-huβ-Gluc fusion protein is thatfusion protein not bound to the tumor is removed from the plasma and thenormal tissues by internalization mainly by receptors for mannose6-phosphate and galactose. (Evidence for this statement is that there isan intracellular increase in the β-glucuronidase level, for example inthe liver).

[0029] As shown in Table 5, the sFv-huβ-Gluc contains relatively largeamounts of galactose and, especially, mannose, which are mainlyresponsible for the binding to the particular receptors. The fusionprotein/receptor complex which results and in which there is binding viathe carbohydrate residues of the fusion protein is then removed from theextracellular compartment by internalization.

[0030] This rapid internalization mechanism, which is mainly mediated bygalactose and mannose, is closely involved in the advantageouspharmacokinetics of the fusion protein according to the invention. Theseadvantageous pharmacokinetics of the fusion protein to which galactoseand, in particular, mannose are attached makes it possible for ahydrophilic prodrug which undergoes extracellular distribution to beadministered i.v. at a relatively early time without elicitingnon-specific prodrug activation. In this case an elimination step asdescribed by Sharma et al. (Brit. J. Cancer, 61, 659, 1990) isunnecessary. Based on the data in Table 4, injection of a suitableprodrug (S. Adrianomenjanahari et al. 1992, Int. Carbohydrate Symp.,Parts A264, 299) is possible even 3 days after injection of thesFv-huβ-Gluc fusion protein without producing significant side effects(data not shown).

[0031] A similarly advantageous attachment of carbohydrates to fusionproteins can also be achieved, for example, by secretory expression ofthe sFv-huβ-Gluc fusion protein in particular yeast strains such asSaccharomyces cerevisiae or Hansenula polymorpha. These organisms arecapable of very effective mannosylation of fusion proteins which haveappropriate N-glycosylation sites (Goochee et al., Biotechnology, 9,1347-1354, 1991). Such fusion proteins which have undergone secretoryexpression in yeast cells show a high degree of mannosylation andfavorable pharmacokinetics comparable to those of the sFv-huβ-Glucfusion protein expressed in BHK cells (data not shown). In this case,the absence of galactose is compensated by the even higher degree ofmannosylation of the fusion protein (Table 6). The sFv-huβ-Gluc fusionprotein described above was constructed by genetic manipulation andexpressed in yeast as described in detail in Example 9.

[0032] Instead of human β-glucuronidase it is, however, also possible toemploy another glucuronidase with advantageous properties. For example,the E.coli β-glucuronidase has the particular advantage that itscatalytic activity at pH 7.4 is significantly higher than that of humanβ-glucuronidase. In Example 10, an sFv-E.coli β-Gluc construct wasprepared by methods of genetic manipulation and underwent secretoryexpression as functionally active mannosylated fusion protein inSaccharomyces cerevisiae. The pharmacokinetic data are comparable tothose of the sFv-huβ-Gluc molecule which was expressed in yeast or inBHK cells (Table 4).

[0033] The glucuronidases from the fungus Kobayasia nipponica and fromthe plants Secale cereale have the advantage, for example, that they arealso active as monomers. In Example 11, methods of genetic manipulationwere used to prepare a construct which, after expression inSaccharomyces cerevisiae, excretes an sFv-B. cereus β-lactamase IIfusion protein preferentially in mannosylated form.

[0034] This fusion protein likewise has, as the fusion proteinsaccording to the invention, on the basis of β-glucuronidasepharmacokinetics which are favorable for prodrug activation (Table 4).

[0035] Furthermore, the compounds according to the invention can beemployed not only in combination with a prodrug but also in theframework of conventional chemotherapy in which cytostatics which aremetabolized as glucuronides and thus inactivated can be converted backinto their toxic form by the administered compounds.

[0036] The following examples now describe the synthesis by geneticmanipulation of sFv-β-Gluc fusion proteins, and the demonstration of theability to function.

[0037] The starting material comprised the plasmids pABstop 431/26 humV_(H) and pABstop 431/26 hum VH_(L). These plasmids contain thehumanized version of the V_(H) gene and V_(L) gene of anti-CEA MAb BW431/26 (Gussow and Seemann, 1991, Meth. Enzymology, 203, 99-121).Further starting material comprised the plasmid pABstop 431/26V_(H)-huβ-Gluc 1H (EP-A2-0 501 215) which contains a V_(H) exon,including the V_(H)-intrinsic signal sequence, followed by a CH1 exon,by the hinge exon of a human IgG3 C gene and the complete cDNA of humanβ-glucuronidase.

EXAMPLE 1

[0038] Amplification of the V_(H) and V_(L) Genes of MAb hum 431/26

[0039] The oligonucleotides pAB-Back and linker-anti (Tab. 2) are usedto amplify the V_(H) gene including the signal sequence intrinsic to theV_(H) gene from pABstop 431V_(H) hum (V_(H) 431/26) (Güssow and Seemann,1991, Meth. Enzymology, 203, 99-121). The oligonucleotides linker-senseand V_(L(Mut))-For (Tab. 3) are used to amplify the V_(L) gene frompABstop 431V_(L) hum (V_(L) 431/26).

EXAMPLE 2

[0040] Joining of the V_(H) 431/26 and V_(L) 431/26 Gene Fragments

[0041] The oligonucleotides linker-anti and linker-sense are partiallycomplementary with one another and encode a polypeptide linker which isintended to link the V_(H) domain and V_(L) domain to give an sFvfragment. In order to fuse the amplified V_(H) fragments with the V_(L)fragments, they are purified and employed in a 10-cycle reaction asfollows: H₂O: 37.5 μl dNTPs (2.5 mM): 5.0 μl PCR buffer (10×): 5.0 μlTaq polymerase (Perkin-Elmer Corp., Emmeryville, CA) (2.5 U/μl): 0.5 μl0.5 μg/μl DNA of the V_(L) frag.: 1.0 μl 0.5 μg/μl DNA of the V_(H)frag.: 1.0 μl PCR buffer (10×): 100 mM tris, pH 8.3, 500 mM KCl, 15 mMMgCl2, 0.1% (w/v) gelatin.

[0042] The surface of the reaction mixture is sealed with paraffin, andsubsequently the 10-cycle reaction is carried out in a PCR apparatusprogrammed for 94° C., 1 min; 55° C., 1 min; 72° C., 2 min. 2.5 pmol ofthe flanking primer pAB-Back and V_(L(Mut))-For are added, and a further20 cycles are carried out. The resulting PCR fragment is composed of theV_(H) gene which is linked to the V_(L) gene via a linker. The signalsequence intrinsic to the V_(H) gene is also present in front of theV_(H) gene. The oligonucleotide V_(L(Mut))-For also results in the lastnucleotide base of the V_(L) gene, a C, being replaced by a G. This PCRfragment codes for a humanized single-chain Fv (sFv 431/26).

EXAMPLE 3

[0043] Cloning of the sFv 431/26 Fragment into the Expression VectorWhich Contains the huβ-glucuronidase Gene.

[0044] The sFv fragment from (2) is cut with HindIII and BamHI andligated into the vector pAB 431V_(H) hum/CH1+1h/β-Glc which has beencompletely cleaved with HindIII and partially cleaved with BglII. Thevector pABstop 431/26V_(H)huβ-Gluc1H contains a V_(H) exon, includingthe V_(H)-intrinsic signal sequence, followed by a CH1 exon, by thehinge exon of a human IgG3 C gene and by the complete cDNA of humanβ-glucuronidase. The plasmid clone pMCG-E1 which contains the humanizedsFv 431/26, a hinge exon and the gene for human β-glucuronidase isisolated (pMCG-E1).

EXAMPLE 4

[0045] Expression of the sFv-huβ-Gluc Fusion Protein in BHK Cells.

[0046] The clone pMCG-E1 is transfected with the plasmid pRMH 140 whichharbors a neomycin-resistance gene and with the plasmid pSV2 whichharbors a methotrexateresistance gene into BHK cells. The BHK cellssubsequently express a fusion protein which has both the antigen-bindingproperties of MAb BW 431/26hum and the enzymatic activity of humanβ-glucuronidase.

EXAMPLE 5

[0047] Demonstration of the Antigen-binding Properties and of theEnsymatic Activity of the sFv-huβ-Gluc Fusion Protein.

[0048] The ability of the sFv-huβ-Gluc fusion protein to bindspecifically to the CEA epitope defined by 431/26 and simultaneously toexert the enzymatic activity of human β-glucuronidase was shown in aspecificity enzyme activity test (EP-A2-0 501 215). The test determinesthe liberation of 4-methylumbelliferone from 4-methylumbelliferylβ-glucuronide by the β-glucuronidase portion of the fusion protein afterthe fusion protein has been bound via the sFv portion to an antigen. Themeasured fluorescence values are reported as relative fluorescence units(FU). The test shows a significant liberation of methyl-umbelliferone bythe fusion protein in the plates coated with CEA. By contrast, thefusion protein does not liberate any methylumbelliferone in controlplates coated with PEM (polymorphic epithelial mucin).

EXAMPLE 6

[0049] TSK 3000 Gel Chromatography

[0050] 200 ng of the sFv-huβ-Gluc fusion protein which had been purifiedby anti-idiotype affinity chromatography in 25 μl were chromatographedon a TSK gel G 3000 SW XL column (TOSO HAAS Order No. 3.5Wx N3211, 7.8mm×300 mm) in a suitable mobile phase (PBS, pH 7.2, containing 5 g/lmaltose and 4.2 g/l arginine) at a flow rate of 0.5 ml/ min. The MerckHitachi HPLC system (L-4000 UV detector, L-6210 intelligent pump, D-2500Chromato-integrator) was operated under ≈20 bar, the optical density ofthe eluate was determined at 280 nm, and an LKB 2111 Multisac fractioncollector was used to collect 0.5 ml fractions which were subsequentlyanalysed in a specificity enzyme activity test (SEAT) (EP 0 501 215 A2,Example J). The result of this experiment is shown in FIG. 1. It isclearly evident that the position of the peak detectable by measurementof the optical density at 280 nm coincides with the peak whichdetermines the specificity and enzyme activity (SEAT) of the eluate.Based on the positions of the molecular weights of standard proteinswhich are indicated by arrows, it can be concluded that the functionallyactive sFv-huβ-Gluc fusion protein has an approximate molecular weightof ≈200 kDa under native conditions.

EXAMPLE 7

[0051] Workup of Organs/Tumors for Determination of the Fusion Protein

[0052] The following sequential steps were carried out:

[0053] nude mice (CD1) which have a subcutaneous tumor and have beentreated with fusion protein or antibody-enzyme conjugate undergoretroorbital exsanguination and are then sacrificed

[0054] the blood is immediately placed in an Eppendorf tube whichalready contains 10 μl of Liquemin 25000 (from Hoffman-LaRoche AG)

[0055] centrifugation is then carried out in a centrifuge (Megafuge 1.0,from Heraeus) at 2500 rpm for 10 min

[0056] the plasma is then obtained and frozen until tested

[0057] the organs or the tumor are removed and weighed

[0058] they are then completely homogenized with 2 ml of 1% BSA in PBS,pH 7.2

[0059] the tumor homogenates are adjusted to pH 4.2 with 0.1 N HCl (thesample must not be overtitrated because β-glucuronidase is inactivatedat pH<3.8)

[0060] all the homogenates are centrifuged at 16000 g for 30 min

[0061] the clear supernatant is removed

[0062] the tumor supernatants are neutralized with 0.1 N NaOH

[0063] the supernatants and the plasma can now be quantified inimmunological tests.

EXAMPLE 8

[0064] Triple Determinant Test

[0065] The tests are carried out as follows:

[0066] 75 μl of a mouse anti-huβ-Gluc antibody (MAb 2118/157Behringwerke) diluted to 2 μg/ml in PBS, pH 7.2, are placed in each wellof a microtiter plate (polystyrene U-shape, type B, from Nunc, Order No.4-60445)

[0067] the microtiter plates are covered and incubated at. R.T.overnight

[0068] the microtiter plates are subsequently washed 3× with 250 μl of0.05 M tris-citrate buffer, pH 7.4, per well

[0069] these microtiter plates coated in this way are incubated with 250μl of blocking solution (1% casein in PBS, pH 7.2) in each well at R.T.for 30′ (blocking of non-specific binding sites) (coated microtiterplates which are not required are dried at R.T. for 24 hours and thensealed together with drying cartridges in coated aluminum bags forlong-term storage)

[0070] during the blocking, in an untreated 96-well U-shaped microtiterplate (polystyrene, from Renner, Order No. 12058), 10 samples+2 positivecontrols+1 negative control are diluted 1:2 in 1% casein in PBS, pH 7.2,in 8 stages (starting from 150 μl of sample, 75 μl of sample arepipetted into 75 μl of casein solution etc.)

[0071] the blocking solution is aspirated out of the microtiter platecoated with anti-huβ-Gluc anti-bodies, and 50 μl of the diluted samplesare transferred per well from the dilution plate to the test plate andincubated at R.T. for 30 min

[0072] during the sample incubation, the ABC-AP reagent (fromVectastain, Order No. AK-5000) is made up: thoroughly mix 2 drops ofreagent A (Avidin DH) in 10 ml of 1% casein in PBS, pH 7.2, add 2 dropsof reagent B (biotinylated alkaline phosphatase) add mix thoroughly.(The ABC-AP solution must be made up at least 30′ before use.)

[0073] the test plate is washed 3 times with ELISA washing buffer(Behringwerke, Order No. OSEW 96)

[0074] 50 μl of biotin-labeled detecting antibody mixture (1+1 mixtureof mouse anti 431/26 antibody (MAb 2064/353, Behringwerke) and mouseanti-CEA antibody (MAb 250/183, Behringwerke) in a concentration of 5μg/ml diluted in 1% casein in PBS, pH 7.2, final concentration of eachantibody of 2.5 μg/ml) are placed in each well

[0075] the test plate is washed 3 times with ELISA washing buffer

[0076] 50 μl of the prepared ABC-AP solution are placed in each well andincubated at R.T. for 30 min

[0077] during the ABC-AP incubation, the substrate is made up (freshsubstrate for each test: 1 mM 4-methylumbelliferyl phosphate, Order No.M-8883, from Sigma, in 0.5 M tris +0.01% MgCl₂, pH 9.6)

[0078] the test plate is washed 7 times with ELISA washing buffer

[0079] 50 μl of substrate are loaded into each well, and the test plateis covered and incubated at 37° C. for 2 h

[0080] 150 μl of stop solution (0.2 M glycine+0.2% SDS, pH 11.7) aresubsequently added to each well

[0081] the fluorometric evaluation is carried out in a Fluoroscan II(ICN Biomedicals, Cat.No. 78-611-00) with an excitation wavelength of355 nm and an emission wavelength of 460 nm

[0082] the unknown concentration of fusion protein in the sample isdetermined on the basis of the fluorescence values for the positivecontrol included in the identical experiment (dilution series withpurified sFv-huβ-Gluc mixed with CEA 5 μg/ml as calibration plot).

EXAMPLE 9

[0083] Expression of the sFv-huβ-Gluc Fusion Protein in Yeast.

[0084] The single-chain Fv (sFv) from Example 2 is amplified with theoligos 2577 and 2561 (Table 7) and cloned into the vector pUC19 whichhas been digested with XbaI/HindIII (FIG. 2).

[0085] The human β-glucuronidase gene is amplified with the oligos 2562and 2540 (Table 8) from the plasmid pAB 431/26 V_(H)hum/CH1+1H/P-Gluc(Example 3) and ligated into the plasmid sFv 431/26 in pUC19 (FIG. 2)cut with BglII/HindIII (FIG. 3).

[0086] A KpnI/NcoI fragment is amplified with the oligos 2587 and 2627(Table 9) from the sFv 431/26 and cloned into the yeast expressionvector pIXY digested with KpnI/NcoI (FIG. 4).

[0087] The BstEII/HindIII fragment from the plasmid sFv 431/26 huβ-Glucin pUC19 (FIG. 3) is ligated into the vector PIXY 120 which harbors theV_(H) gene, the linker and a part of the V_(L) gene (V_(H)/link/V_(K)part. in pIXY 120) and has been digested with BstEII/partially withHindIII (FIG. 5).

[0088] The resulting plasmid sFv 431/26 huβ-Gluc in pIXY 120 istransformed into Saccharomyces cerevisiae and the fusion protein isexpressed.

EXAMPLE 10

[0089] Expression of the sFv-E.coli-β-glucuronidase Fusion Protein inYeast.

[0090] The E.coli glucuronidase gene is amplified from PRAJ 275(Jefferson et al. Proc. Natl. Acad. Sci, USA, 83: 8447-8451, 1986) withthe oligos 2638 and 2639 (Table 10) and ligated into sFv 431/26 in pUC19(Example 9, FIG. 2) cut with BglII/HindIII (FIG. 6).

[0091] A BstEII/HindIII fragment from sFv 431/26 E.coli β-Gluc in pUC19is cloned into the vector V_(H)/link/V_(K) part in PIXY 120 (Example 9,FIG. 4) which has been partially digested with BstEII/HindIII (FIG. 7).

[0092] The plasmid sFv 431/26 E.coli β-Gluc in pIXY 120 is transformedinto Saccharomyces cerevisiae and the fusion protein is expressed.

EXAMPLE 11

[0093] Expression of the sFv-β-lactamase Fusion Protein in Yeast.

[0094] The single-chain Fv (sFv) from Example 2 is amplified with theoligos 2587 and 2669 (Table 11) and ligated into the pUC19 vectordigested with KpnI/HindIII (FIG. 8).

[0095] The β-lactamase II gene (Hussain et al., J. Bacteriol. 164:223-229, 1985) is amplified with the oligos 2673 and 2674 (Table 11)from the complete DNA of Bacillus cereus and ligated into the pUC19vector digested with EcoRI/HindIII (FIG. 9). A BclI/HindIII fragment ofthe β-lactamase gene is ligated into sFv 431/26 in pUC19 which has beencut with BglII/HindIII (FIG. 10).

[0096] The KpnI/HindIII sFv-β-lactamase fragment is ligated into pIXY120 which has been digested with KpnI/partially with HindIII (FIG. 11).The plasmid is transformed into Saccharomyces cerevisiae, and a fusionprotein which has both the antigen-binding properties of MAb 431/26 andthe enzymatic activity of Bacillus cereus β-lactamase is expressed.TABLE 1 CCAAGCTTAT GAATATGCAA ATCCTGCTCA TGAATATGCA AATCCTCTGA 50ATCTACATGG TAAATATAGG TTTGTCTATA CCACAAACAG AAAAACATGA 100 GATCACAGTTCTCTCTACAG TTACTGAGCA CACAGGACCT CACC ATG GGA TGG 153                                                 Met Gly Trp AGC TGT ATCATC CTC TTC TTG GTA GCA ACA GCT ACA GGTAAGGGGC 199 Ser Cys Ile Ile LeuPhe Leu Val Ala Thr Ala Thr -10 TCACAGTAGC AGGCTTGAGG TCTGGACATATATATGGGTG ACAATGACAT 249 CCACTTTGCC TTTCTCTCCA CA GGT GTC CAC TCC CAGGTC CAA CTG CAG 298                          Gly Val His Ser Gln Val GlnLeu Gln                                          1 GAG AGC GGT CCA GGTCTT GTG AGA CCT AGC CAG ACC CTG AGC CTG 343 Glu Ser Gly Pro Gly Leu ValArg Pro Ser Gln Thr Leu Ser Leu                 10                                      20 ACC TGC ACCGTG TCT GGC TTC ACC ATC AGC AGT GGT TAT AGC TGG 388 Thr Cys Thr Val SerGly Phe Thr Ile Ser Ser Gly Tyr Ser Trp                                     30 CAC TGG GTG AGA CAG CCA CCT GGACGA GGT CTT GAG TGG ATT GGA 433 His Trp Val Arg Gln Pro Pro Gly Arg GlyLeu Glu Trp Ile Gly                 40                                      50 TAC ATA CAGTAC AGT GGT ATC ACT AAC TAC AAC CCC TCT CTC AAA 478 Tyr Ile Gln Tyr SerGly Ile Thr Asn Tyr Asn Pro Ser Leu Lys                                     60 AGT AGA GTG ACA ATG CTG GTA GACACC AGC AAG AAC CAG TTC AGC 523 Ser Arg Val Thr Met Leu Val Asp Thr SerLys Asn Gln Phe Ser                 70                                      80 CTG AGA CTCAGC AGC GTG ACA GCC GCC GAC ACC GCG GTC TAT TAT 568 Leu Arg Leu Ser SerVal Thr Ala Ala Asp Thr Ala Val Tyr Tyr                                     90 TGT GCA AGA GAA GAC TAT GAT TACCAC TGG TAC TTC GAT GTC TGG 613 Cys Ala Arg Glu Asp Tyr Asp Tyr His TrpTyr Phe Asp Val Trp                100                                     110 GGC CAA GGGACC ACG GTC ACC GTC TCC TCA GGA GGC GGT GGA TCG 658 Gly Gln Gly Thr ThrVal Thr Val Ser Ser Gly Gly Gly Gly Ser                                    120 GGC GGT GGT GGG TCG GGT GGC GGCGGA TCT GAC ATC CAG CTG ACC 703 Gly Gly Gly Gly Ser Gly Gly Gly Gly SerAsp Ile Gln Leu Thr                130                                     140 CAG AGC CCAAGC AGC CTG AGC GCC AGC GTG GGT GAC AGA GTG ACC 748 Gln Ser Pro Ser SerLeu Ser Ala Ser Val Gly Asp Arg Val Thr                                    150 ATC ACC TGT AGT ACC AGC TCG AGTGTA AGT TAC ATG CAC TGG TAC 793 Ile Thr Cys Ser Thr Ser Ser Ser Val SerTyr Met His Trp Tyr                160                                     170 CAG CAG AAGCCA GGT AAG GCT CCA AAG CTG CTG ATC TAC AGC ACA 838 Gln Gln Lys Pro GlyLys Ala Pro Lys Leu Leu Ile Tyr Ser Thr                                    180 TCC AAC CTG GCT TCT GGT GTG CCAAGC AGA TTC AGC GGT AGC GGT 883 Ser Asn Leu Ala Ser Gly Val Pro Ser ArgPhe Ser Gly Ser Gly                190                                     200 AGC GGT ACCGAC TTC ACC TTC ACC ATC AGC AGC CTC CAG CCA GAG 928 Ser Gly Thr Asp PheThr Phe Thr Ile Ser Ser Leu Gln Pro Glu                                    210 GAC ATC GCC ACC TAC TAC TGC CATCAG TGG AGT AGT TAT CCC ACG 973 Asp Ile Ala Thr Tyr Tyr Cys His Gln TrpSer Ser Tyr Pro Thr                220                                     230 TTC GGC CAAGGG ACC AAG CTG GAG ATC AAA GGTGAGTAGA ATTTAAACTT 1023 Phe Gly Gln GlyThr Lys Leu Glu Ile Lys                                     240TGCTTCCTCA GTTGGATCTG AGTAACTCCC AATCTTCTCT CTGCA GAG CTC AAA 1077                                                  Glu Leu Lys ACC CCACTT GGT GAC ACA ACT CAC ACA TGC CCA CGG TGC CCA 1119 Thr Pro Leu Gly AspThr Thr His Thr Cys Pro Arg Cys Pro                         250GGTAAGCCAG CCCAGGACTC GCCCTCCAGC TCAAGGCGGG ACAAGAGCCC 1169 TAGAGTGGCCTGAGTCCAGG GACAGGCCCC AGCAGGGTGC TGACGCATCC 1219 ACCTCCATCC CAGATCCCCGTAACTCCCAA TCTTCTCTCT GCA GCG GCG GCG 1271                                                Ala Ala Ala                                                         260 GCG GTG CAGGGC GGG ATG CTG TAC CCC CAG GAG AGC CCG TCG CGG 1316 Ala Val Gln Gly GlyMet Leu Tyr Pro Gln Glu Ser Pro Ser Arg                                    270 GAG TGC AAG GAG CTG GAC GGC CTCTGG AGC TTC CGC GCC GAC TTC 1361 Glu Cys Lys Glu Leu Asp Gly Leu Trp SerPhe Arg Ala Asp Phe                280                                     290 TCT GAC AACCGA CGC CGG GGC TTC GAG GAG CAG TGG TAC CGG CGG 1406 Ser Asp Asn Arg ArgArg Gly Phe Glu Glu Gln Trp Tyr Arg Arg                                    300 CCG CTG TGG GAG TCA GGC CCC ACCGTG GAC ATG CCA GTT CCC TCC 1451 Pro Leu Trp Glu Ser Gly Pro Thr Val AspMet Pro Val Pro Ser                310                                     320 AGC TTC AATGAC ATC AGC CAG GAC TGG CGT CTG CGG CAT TTT GTC 1496 Ser Phe Asn Asp IleSer Gln Asp Trp Arg Leu Arg His Phe Val                                    330 GGC TGG GTG TGG TAC GAA CGG GAGGTG ATC CTG CCG GAG CGA TGG 1541 Gly Trp Val Trp Tyr Glu Arg Glu Val IleLeu Pro Glu Arg Trp                340                                     350 ACC CAG GACCTG CGC ACA AGA GTG GTG CTG AGG ATT GGC AGT GCC 1586 Thr Gln Asp Leu ArgThr Arg Val Val Leu Arg Ile Gly Ser Ala                                    360 CAT TCC TAT GCC ATC GTG TGG GTGAAT GGG GTC GAC ACG CTA GAG 1631 His Ser Tyr Ala Ile Val Trp Val Asn GlyVal Asp Thr Leu Glu                370                                     380 CAT GAG GGGGGC TAC CTC CCC TTC GAG GCC GAC ATC AGC AAC CTG 1676 His Glu Gly Gly TyrLeu Pro Phe Glu Ala Asp Ile Ser Asn Leu                                    390 GTC CAG GTG GGG CCC CTG CCC TCCCGG CTC CGA ATC ACT ATC GCC 1721 Val Gln Val Gly Pro Leu Pro Ser Arg LeuArg Ile Thr Ile Ala                400                                     410 ATC AAC AACACA CTC ACC CCC ACC ACC CTG CCA CCA GGG ACC ATC 1766 Ile Asn Asn Thr LeuThr Pro Thr Thr Leu Pro Pro Gly Thr Ile                                    420 CAA TAC CTG ACT GAC ACC TCC AAGTAT CCC AAG GGT TAC TTT GTC 1811 Gln Tyr Leu Thr Asp Thr Ser Lys Tyr ProLys Gly Tyr Phe Val                430                                     440 CAG AAC ACATAT TTT GAC TTT TTC AAC TAC GCT GGA CTG CAG CGG 1856 Gln Asn Thr Tyr PheAsp Phe Phe Asn Tyr Ala Gly Leu Gln Arg                                    450 TCT GTA CTT CTG TAC ACG ACA CCCACC ACC TAC ATC GAT GAC ATC 1901 Ser Val Leu Leu Tyr Thr Thr Pro Thr ThrTyr Ile Asp Asp Ile                460                                     470 ACC GTC ACCACC AGC GTG GAG CAA GAC AGT GGG CTG GTG AAT TAC 1946 Thr Val Thr Thr SerVal Glu Gln Asp Ser Gly Leu Val Asn Tyr                                    480 CAG ATC TCT GTC AAG GGC AGT AACCTG TTC AAG TTG GAA GTG CGT 1991 Gln Ile Ser Val Lys Gly Ser Asn Leu PheLys Leu Glu Val Arg                490                                     500 CTT TTG GATGCA GAA AAC AAA GTC GTG GCG AAT GGG ACT GGG ACC 2036 Leu Leu Asp Ala GluAsn Lys Val Val Ala Asn Gly Thr Gly Thr                                    510 CAG GGC CAA CTT AAG GTG CCA GGTGTC AGC CTC TGG TGG CCG TAC 2081 Gln Gly Gln Leu Lys Val Pro Gly Val SerLeu Trp Trp Pro Tyr                520                                     530 CTG ATG CACGAA CGC CCT GCC TAT CTG TAT TCA TTG GAG GTG CAG 2126 Leu Met His Glu ArgPro Ala Tyr Leu Tyr Ser Leu Glu Val Gln                                    540 CTG ACT GCA CAG ACG TCA CTG GGGCCT GTG TCT GAC TTC TAC ACA 2171 Leu Thr Ala Gln Thr Ser Leu Gly Pro ValSer Asp Phe Tyr Thr                550                                     560 CTC CCT GTGGGG ATC CGC ACT GTG GCT GTC ACC AAG AGC CAG TTC 2216 Leu Pro Val Gly IleArg Thr Val Ala Val Thr Lys Ser Gln Phe                                    570 CTC ATC AAT GGG AAA CCT TTC TATTTC CAC GGT GTC AAC AAG CAT 2261 Leu Ile Asn Gly Lys Pro Phe Tyr Phe HisGly Val Asn Lys His                580                                     590 GAG GAT GCGGAC ATC CGA GGG AAG GGC TTC GAC TGG CCG CTG CTG 2306 Glu Asp Ala Asp IleArg Gly Lys Gly Phe Asp Trp Pro Leu Leu                                    600 GTG AAG GAC TTC AAC CTG CTT CGCTGG CTT GGT GCC AAC GCT TTC 2351 Val Lys Asp Phe Asn Leu Leu Arg Trp LeuGly Ala Asn Ala Phe                610                                     620 CGT ACC AGCCAC TAC CCC TAT GCA GAG GAA GTG ATG CAG ATG TGT 2396 Arg Thr Ser His TyrPro Tyr Ala Glu Glu Val Met Gln Met Cys                                    630 GAC CGC TAT GGG ATT GTG GTC ATCGAT GAG TGT CCC GGC GTG GGC 2441 Asp Arg Tyr Gly Ile Val Val Ile Asp GluCys Pro Gly Val Gly                640                                     650 CTG GCC CTGCCG CAG TTC TTC AAC AAC GTT TCT CTG CAT CAC CAC 2486 Leu Ala Leu Pro GlnPhe Phe Asn Asn Val Ser Leu His His His                                    660 ATG CAG GTG ATG GAA GAA GTG GTGCGT AGG GAC AAG AAC CAC CCC 2531 Met Gln Val Met Glu Glu Val Val Arg ArgAsp Lys Asn His Pro                670                                     680 GCG GTC GTGATG TGG TCT GTG GCC AAC GAG CCT GCG TCC CAC CTA 2576 Ala Val Val Met TrpSer Val Ala Asn Glu Pro Ala Ser His Leu                                    690 GAA TCT GCT GGC TAC TAC TTG AAGATG GTG ATC GCT CAC ACC AAA 2621 Glu Ser Ala Gly Tyr Tyr Leu Lys Met ValIle Ala His Thr Lys                700                                     710 TCC TTG GACCCC TCC CGG CCT GTG ACC TTT GTG AGC AAC TCT AAC 2666 Ser Leu Asp Pro SerArg Pro Val Thr Phe Val Ser Asn Ser Asn                                    720 TAT GCA GCA GAC AAG GGG GCT CCGTAT GTG GAT GTG ATC TGT TTG 2711 Tyr Ala Ala Asp Lys Gly Ala Pro Tyr ValAsp Val Ile Cys Leu                730                                     740 AAC AGC TACTAC TCT TGG TAT CAC GAC TAC GGC CAC CTG GAG TTG 2756 Asn Ser Tyr Tyr SerTrp Tyr His Asp Tyr Gly His Leu Glu Leu                                    750 ATT CAG CTG CAG CTG GCC ACC CAGTTT GAG AAC TGG TAT AAG AAC 2801 Ile Gln Leu Gln Leu Ala Thr Gln Phe GluAsn Trp Tyr Lys Lys                760                                     770 TAT CAG AAGCCC ATT ATT CAG AGC GAG TAT GGA GCA GAA ACG ATT 2846 Tyr Gln Lys Pro IleIle Gln Ser Glu Tyr Gly Ala Glu Thr Ile                                    780 GCA GGG TTT CAC CAG GAT CCA CCTCTG ATG TTC ACT GAA GAG TAC 2891 Ala Gly Phe His Gln Asp Pro Pro Leu MetPhe Thr Glu Glu Tyr                790                                     800 GAG AAA AGTCTG CTA GAG CAG TAC CAT CTG GGT CTG GAT CAA AAA 2936 Gln Lys Ser Leu LeuGlu Gln Tyr His Leu Gly Leu Asp Gln Lys                                    810 CGC AGA AAA TAT GTG GTT GGA GAGCTC ATT TGG AAT TTT GCC GAT 2981 Arg Arg Lys Tyr Val Val Gly Glu Leu IleTrp Asn Phe Ala Asp                820                                     830 TTC ATC ACTGAA CAG TCA CCG ACG AGA GTG CTG GGG ATT AAA AAG 3026 Phe Met Thr Glu GlnSer Pro Thr Arg Val Leu Gly Asn Lys Lys                                    840 GGG ATC TTC ACT CGG CAG AGA CAACCA AAA AGT GCA GCG TTC CTT 3071 Gly Ile Phe Thr Arg Gln Arg Gln Pro LysSer Ala Ala Phe Leu                850                                     860 TTG CGA GAGAGA TAC TGG AAG ATT GCC AAT GAA ACC AGG TAT CCC 3116 Leu Arg Glu Arg TyrTrp Lys Ile Ala Asn Glu Thr Arg Tyr Pro                                    870 CAC TCA GTA GCC AAG TCA CAA TGTTTG GAA AAC AGC CCG TTT ACT 3161 His Ser Val Ala Lys Ser Gln Cys Leu GluAsn Ser Pro Phe Thr                880                                     890 TGAGCAAGACTGA TACCACCTGC GTGTCCCTTC CTCCCCGAGT CAGGGCGACT 3214 . . .TCCACAGCAG CAGAACAAGT GCCTCCTGGA CTGTTCACGG CAGACCAGAA 3264 CGTTTCTGGCCTGGGTTTTG TGGTCATCTA TTCTAGCAGG GAACACTAAA 3314

[0097] TABLE 2 pAB-Back: 5′                               3′ ACC AGA AGCTTA TGA ATA TGC AAA TC′ Linker-Anti: 5′ GCC ACC CGA CCC ACC ACC GCC CGATCC ACC GCC TCC                            3′ TGA GGA GAC GGT GAC CGTGGT C

[0098] TABLE 3 Linker-Sense: 5′ GGT GGA TCG GGC GGT GGT GGG TCG GGT GGCGGC GGA                              3′ TCT GAC ATC CAG CTG ACC CAG AGCVL(Mut)-For: 5′ TGC AGG ATC CAA CTG AGG AAG CAA AGT TTA AAT TCT                3′ ACT CAC CTT TGA TC

[0099] TABLE 4 Pharmacokinetics of sFv-hu β Gluc fusion protein in CDlnu/nu mice carrying MzStol ng of sFv-huβGluc per gram of tissue or ml ofplasma measured in the triple determinant test Mouse 1 Mouse 2 Mouse 3Mouse 4 Mouse 5a Mouse 5b Tissue type 0.05 h 3 h 24 h 48 h 120 h 120 hTumor 24.8 4 7.7 2.1 2.2 6.2 Spleen 15.4 4.1 <0.1 <0.1 <0.1 <0.1 Liver40.9 10.1 0.8 0.8 0.3 <0.1 Intestine 5.2 4.4 1.1 1.2 0.6 <0.1 Kidney44.4 7 <0.1 <0.1 <0.1 <0.1 Lung 154.8 17.3 <0.1 <0.1 <0.1 <0.1 Heart148.3 8.2 <0.1 <0.1 <0.1 <0.1 Plasma 630.9 95 2.7 0.4 <0.1 <0.1

[0100] TABLE 5 Analysis of the monosaccharide components in thecarbohydrate content of the sFv-huB-Gluc fusion protein from BHK cellsThe purified sFv-huB-Gluc fusion protein was investigated for itscarbohydrate content. This revealed after hydrolysis the followingindividual components in the stated molar ratio (mol of carbohydrate/molof sFv-huB-Gluc). N-Acetyl N-Acetyl- Fucose Galactosamine glucosamineGalactose Glucose Mannose neuraminic acid sFv-huB-Gluc 4 2 30 8 1 43 4

[0101] Methods:

[0102] Neuraminic acid was determined by the method of Hermentin andSeidat (1991) GBF Monographs Volume 15, pp. 185-188 (after hydrolysisfor 30 min in the presence of 0.1 N sulfuric acid at 80° C. andsubsequent neutralization with 0.4 N sodium hydroxide solution) byhigh-pH anion exchange chromatography with pulsed amerometric detection(HPAE-PAD).

[0103] The monosaccaride components were determined (after hydrolysisfor 4 h in the presence of 2 N trifluoracetic acid at 100° C. andevaporation to dryness in a SpeedVac) likewise by HPAE-PAD in amotivation of the method described by Hardy et al. (1988) AnalyticalBiochemistry 170, pp. 54-62. TABLE 6 Analysis of the monosaccharidecomponents in the carbohydrate content of the sFv-huβGluc fusion proteinfrom Saccharomyces cerevisiae. Glucosamine Glucose Mannose sFv-huβGluc 612 150 mol/mol (mol/mol)

[0104] TABLE 7 Oligos for sFv 431/26 cloning in pUC 19 sFv for (2561) 5′TTT TTA AGC TTA GAT CTC CAC CTT GGT C 3′ sFv back (2577) 5′ AAAAAT CTA GAA TGC AGG TCC AAC TGC AGG AGA G 3′

[0105] TABLE 8 Oligos for hum.β-Gluc cloning in sFv pUC 19 Hum.β-Gluc.back oligo (2562) 5′ AAA AAA GTG ATC AAA GCG TCT GGC GGG CCA CAG    GGCGGG ATC CTG TAC 3′ Hum.β-Gluc for oligo (2540) 5′ TTT TAA GCT TCA AGTAAA CGG GCT GTT 3′

[0106] TABLE 9 Oligos for sFv/hum-β-Gluc cloning in pIXY120 PCR oligoVHpIXY back (2587) 5′ TTT TGG TAC CTT TGG ATA AAA GAC AGG TCC AAC TGC   AGG AGAG 3′ PCR oligo VKpIXY for (2627) 5′ A AAA CCA TGG GAA TTCAAG CTT CGA GCT GGT ACT    ACA GGT 3′

[0107] TABLE 10 Oligos for E.coli β-Gluc cloning in sFv pUC 19 E. coliβ-Gluc. for (2639) 5′ TTT TAA GCT TCC ATG GCG GCC GCT CAT TGT TTG    CCTCCC TGC TG 3′ E. coli β-Gluc. back (2638) 5′ AAA AAG ATC TCC GCG TCT GGCGGG CCA CAG TTA    CGT GTA GAA ACC CCA 3′

[0108] TABLE 11 Oligos for sFv/β-lactamase cloning in pIXY120 PCR oligoVHpIXY back (2587) 5′ TTT TGG TAC CTT TGG ATA AAA GAC AGG TCC AAC TGC   AGG AGA G 3′ PCR oligo VKpIXYβ-lactamase for (2669) 5′ AAA AAG CTTAGA TCT CCA GCT TGG TCC C 3′ PCR oligo link /β-lactamase back (2673) 5′AAA GAA TTC TGA TCA AAT CCT CGA GCT CAG GTT CAC    AAA AGG TAG AGA AAACAG T 3′ linker PCR oligo β-lactamase for (2674) 5′ TTT AAG CTT ATT TTAATA AAT CCA ATG T 3′

1. A compound containing an antigen binding region which is bound to atleast one prodrug-activating enzyme, where the antigen binding region iscomposed of a single polypeptide chain.
 2. A compound as claimed inclaim 1, wherein the compound carries covalently bonded carbohydrates.3. A compound as claimed in claim 1, wherein the antigen binding regioncontains a variable domain of a heavy antibody chain and a variabledomain of a light antibody chain (sFv fragment).
 4. A compound asclaimed in claim 1, wherein the antigen binding region binds to atumor-associated antigen (TAA).
 5. A compound as claimed in claim 3,wherein the TAA is an N-CAM, PEM, EGF-R, Sialyl-Le^(a), Sialyl-Le^(x),TFβ, GICA, GD₃, GD₂, TAG72, CA125, the 24-25 kDa glycoprotein defined byMAb L6, or CEA, preferably a CEA.
 6. A compound as claimed in claim 1,wherein the enzyme is a lactamase, preferably a Bacillus cereus IIβ-lactamase, pyroglutamate aminopeptidase, D-aminopeptidase, oxidase,peroxidase, phosphatase, hydroxynitrile lyase, protease, esterase,carboxypeptidase, preferably a carboxypeptidase G2 from Pseudomonas orglycosidase.
 7. A compound as claimed in claim 6, wherein the enzyme isa β-glucuronidase, preferably a E.coli, Kobayasia nipponica, Secalecereale or human β-glucuronidase.
 8. A compound as claimed in claim 1,wherein the antigen binding region is linked to the enzyme via a peptidelinker.
 9. A compound as claimed in claim 1, wherein the glycosylationtakes place either by means of chemical methods or by a selection ofsuitable expression systems.
 10. A compound as claimed in claim 1, whichundergoes secretory expression in Saccharomyces cerevisiae or, moreadvantageously, in Hansenula polymorpha.
 11. A compound as claimed inclaim 1, which is expressed in E. coli and is subsequently chemicallyglycosylated, preferably galactosylated and/or mannosylated.
 12. Acompound as claimed in claim 1, wherein the sFv-β-lactamase fusionprotein, which has undergone periplasmic expression in E. coli, ischemically glycosylated, preferably galactosylated and/or mannosylated.13. A compound as claimed in claim 1, wherein the sFv-β-lactamase fusionprotein undergoes secretory expression in Saccharomyces cerevisiae orHansenula polymorpha.
 14. A nucleic acid coding for a compound asclaimed in claim
 1. 15. A nucleic acid as claimed in claim 14, codingfor a humanized sFv fragment against CEA and a human β-glucuronidase.16. A nucleic acid as claimed in claim 14 with the sequence CCAAGCTTATGAATATGCAA ATCCTGCTCA TGAATATGCA AATCCTCTGA 50 ATCTACATGG TAAATATAGGTTTGTCTATA CCACAAACAG AAAAACATGA 100 GATCACAGTT CTCTCTACAG TTACTGAGCACACAGGACCT CACC ATG GGA TGG 153                                                 Met Gly Trp AGC TGT ATCATC CTC TTC TTG GTA GCA ACA GCT ACA GGTAAGGGGC 199 Ser Cys Ile Ile LeuThe Leu Val Ala Thr Ala Thr                         −10 TCACAGTAGCAGGCTTGAGG TCTGGACATA TATATGGGTG ACAATGACAT 249 CCACTTTGCC TTTCTCTCCA CAGGT GTC CAC TCC CAG GTC CAA CTG CAG 298                          Gly ValHis Ser Gln Val Gln Leu Gln                                          1GAG AGC GGT CCA GGT CTT GTG AGA CCT AGC CAG ACC CTC AGC CTG 343 Glu SerGly Pro Gly Leu Val Arg Pro Ser Gln Thr Leu Ser Leu                 10                                      20 ACC TGC ACCGTG TCT GGC TTC ACC ATC AGC AGT GGT TAT AGC TGG 388 Thr Cys Thr Val SerGly Phe Thr Ile Ser Ser Gly Tyr Ser Trp                                     30 CAC TGG GTG AGA CAG CCA CCT GGACGA GGT CTT GAG TGG ATT GGA 433 His Trp Val Arg Gln Pro Pro Gly Arg GlyLeu Glu Trp Ile Gly                 40                                      50 TAC ATA CAGTAC AGT GGT ATC ACT AAC TAC AAC CCC TCT CTC AAA 478 Tyr Ile Gln Tyr SerGly Ile Thr Asn Tyr Asn Pro Ser Leu Lys                                    60 AGT AGA GTG ACA ATG CTG GTA GACACC AGC AAG AAC CAG TTC AGC 523 Ser Arg Val Thr Met Leu Val Asp Thr SerLys Asn Gln Phe Ser                 70                                      80 CTG AGA CTCAGC AGC GTG ACA GCC GCC GAC ACC GCG GTC TAT TAT 568 Leu Arg Leu Ser SerVal Thr Ala Ala Asp Thr Ala Val Tyr Tyr                                     90 TGT GCA AGA GAA GAC TAT GAT TACCAC TGG TAC TTC GAT GTC TGG 613 Cys Ala Arg Glu Asp Tyr Asp Tyr His TrpTyr Phe Asp Val Trp                100                                     110 GGC CAA GGGACC ACG GTC ACC GTC TCC TCA GGA GGC GGT GGA TCG 658 Gly Gln Gly Thr ThrVal Thr Val Ser Ser Gly Gly Gly Gly Ser                                    120 GGC CGT GGT GGG TCG GGT GGC GGCGGA TCT GAC ATC CAG CTG ACC 703 Gly Gly Gly Gly Ser Gly Gly Gly Gly SerAsp Ile Gln Leu Thr                130                                     140 CAG AGC CCAAGC AGC CTG AGC GCC AGC GTG GGT GAC AGA GTG ACC 748 Gln Ser Pro Ser SerLeu Ser Ala Ser Val Gly Asp Arg Val Thr                                    150 ATC ACC TGT AGT ACC AGC TCG AGTGTA AGT TAC ATG CAC TGG TAC 793 Ile Thr Cys Ser Thr Ser Ser Ser Val SerTyr Met His Trp Tyr                160                                     170 CAG CAG AAGCCA GGT AAG GCT CCA AAG CTG CTG ATC TAC AGC ACA 838 Gln Gln Lys Pro GlyLys Ala Pro Lys Leu Leu Ile Tyr Ser Thr                                    180 TCC AAC CTG GCT TCT GGT GTG CCAAGC AGA TTC AGC GGT AGC GGT 883 Ser Asn Leu Ala Ser Gly Val Pro Ser ArgPhe Ser Gly Ser Gly                190                                     200 AGC GGT ACCGAC TTC ACC TTC ACC ATC AGC AGC CTC CAG CCA GAG 928 Ser Gly Thr Asp PheThr Phe Thr Ile Ser Ser Leu Gln Pro Glu                                    210 GAC ATC GCC ACC TAC TAC TGC CATCAG TGG AGT AGT TAT CCC ACG 973 Asp Ile Ala Thr Tyr Tyr Cys His Gln TrpSer Ser Tyr Pro Thr                220                                     230 TTC GGC CAAGGG ACC AAG CTG GAG ATC AAA GGTGAGTAGA ATTTAAACTT 1023 Phe Gly Gln GlyThr Lys Leu Glu Ile Lys                                     240TGCTTCCTCA GTTGGATCTG AGTAACTCCC AATCTTCTCT CTGCA GAG CTC AAA 1077                                                  Glu Leu Lys ACC CCACTT GGT GAC ACA ACT CAC ACA TGC CCA CGG TGC CCA 1119 Thr Pro Leu Gly AspThr Thr His Thr Cys Pro Arg Cys Pro                         250GGTAAGCCAG CCCAGGACTC GCCCTCCAGC TCAAGGCGGG ACAAGAGCCC 1169 TAGAGTGGCCTGAGTCCAGG GACAGGCCCC AGCAGGGTGC TGACGCATCC 1219 ACCTCCATCC CAGATCCCCGTAACTCCCAA TCTTCTCTCT GCA GCG GCG GCG 1271                                               Ala Ala Ala                                                       260 GCG GTG CAGGGC GGG ATG CTG TAC CCC CAG GAG AGC CCG TCG CGG 1316 Ala Val Gln Gly GlyMet Leu Tyr Pro Gln Glu Ser Pro Ser Arg                                    270 GAG TGC AAG GAG CTG GAC GGC CTCTGG AGC TTC CGC GCC GAC TTC 1361 Glu Cys Lys Glu Leu Asp Gly Leu Trp SerPhe Arg Ala Asp Phe                280                                     290 TCT GAC AACCGA CGC CGG GGC TTC GAG GAG CAG TGG TAC CGG CGG 1406 Ser Asp Asn Arg ArgArg Gly Phe Glu Glu Gln Trp Tyr Arg Arg                                    300 CCG CTG TGG GAG TCA GGC CCC ACCGTG GAC ATG CCA GTT CCC TCC 1451 Pro Leu Trp Glu Ser Gly Pro Thr Val AspMet Pro Val Pro Ser                310                                     320 AGC TTC AATGAC ATC AGC CAG GAC TGG CGT CTG CGG CAT TTT GTC 1496 Ser Phe Asn Asp IleSer Gln Asp Trp Arg Leu Arg His Phe Val                                    330 GGC TGG GTG TGG TAC GAA CGG GAGGTG ATC GTG CCG GAG CGA TGG 1541 Gly Trp Val Trp Tyr Glu Arg Glu Val IleLeu Pro Glu Arg Trp                340                                     350 ACC CAG GACCTG CGC ACA AGA GTG GTG CTG AGG ATT GGC AGT GCC 1586 Thr Gln Asp Leu ArgThr Arg Val Val Leu Arg Ile Gly Ser Ala                                    360 CAT TCC TAT GCC ATC GTG TGG GTGAAT GGG GTC GAC ACG CTA GAG 1631 His Ser Tyr Ala Ile Val Trp Val Asn GlyVal Asp Thr Leu Glu                370                                     380 CAT GAG GGGGGC TAC CTC CCC TTC GAG GCC GAC ATC AGC AAC CTG 1676 His Glu Gly Gly TyrLeu Pro Phe Glu Ala Asp Ile Ser Asn Leu                                    390 GTC CAG GTG GGG CCC CTG CCC TCCCGG CTC CGA ATC ACT ATC GCC 1721 Val Gln Val Gly Pro Leu Pro Ser Arg LeuArg Ile Thr Ile Ala                400                                     410 ATC AAC AACACA CTC ACC CCC ACC ACC CTG CCA CCA GGG ACC ATC 1766 Ile Asn Asn Thr LeuThr Pro Thr Thr Leu Pro Pro Gly Thr Ile                                    420 CAA TAC CTG ACT GAC ACC TCC AAGTAT CCC AAG GGT TAC TTT GTC 1811 Gln Tyr Leu Thr Asp Thr Ser Lys Tyr ProLys Gly Tyr Phe Val                430                                     440 CAG AAC ACATAT TTT GAC TTT TTC AAC TAC GCT GGA CTC CAG CGG 1856 Gln Asn Thr Tyr PheAsp Phe Phe Asn Tyr Ala Gly Leu Gln Arg                                    450 TCT GTA CTT CTG TAC ACG ACA CCCACC ACC TAC ATC GAT GAC ATC 1901 Ser Val Leu Leu Tyr Thr Thr Pro Thr ThrTyr Ile Asp Asp Ile                460                                     470 ACC GTC ACCACC ACC GTG GAG CAA GAC AGT GGG CTG GTG AAT TAC 1946 Thr Val Thr Thr SerVal Glu Gln Asp Ser Gly Leu Val Asn Tyr                                    480 CAG ATC TCT GTC AAG GGC AGT AACCTG TTC AAG TTG GAA GTG CGT 1991 Gln Ile Ser Val Lys Gly Ser Asn Leu PheLys Leu Glu Val Arg                490                                     500 CTT TTG GATGCA GAA AAC AAA GTC GTG GCG AAT GGG ACT GGG ACC 2036 Leu Leu Asp Ala GluAsn Lys Val Val Ala Asn Gly Thr Gly Thr                                    510 CAG GGC CAA CTT AAG GTG CCA GGTGTC AGC CTC TGG TGG CCG TAC 2081 Gln Gly Gln Leu Lys Val Pro Gly Val SerLeu Trp Trp Pro Tyr                520                                     530 CTG ATG CACGAA CGC CCT GCC TAT CTG TAT TCA TTG GAG GTG CAG 2126 Leu Met His Glu ArgPro Ala Tyr Leu Tyr Ser Leu Glu Val Gln                                    540 CTG ACT GCA CAG ACG TCA CTG GGGCCT GTG TCT GAC TTC TAC ACA 2171 Leu Thr Ala Gln Thr Ser Leu Gly Pro ValSer Asp Phe Tyr Thr                550                                     560 CTC CCT GTGGGG ATC CGC ACT GTG GCT GTC ACC AAG AGC CAG TTC 2216 Leu Pro Val Gly IleArg Thr Val Ala Val Thr Lys Ser Gln Phe                                    570 CTC ATC AAT GGG AAA CCT TTC TATTTC CAC GGT GTC AAC AAG CAT 2261 Leu Ile Asn Gly Lys Pro Phe Tyr Phe HisGly Val Asn Lys His                580                                     590 GAG GAT GCGGAC ATC CGA GGG AAG GGC TTC GAC TGG CCG CTC CTG 2306 Glu Asp Ala Asp IleArg Gly Lys Gly Phe Asp Trp Pro Leu Leu                                    600 GTG AAG GAC TTC AAC CTG CTT CGCTGG CTT GGT GCC AAC GCT TTC 2351 Val Lys Asp Phe Asn Leu Leu Arg Trp LeuGly Ala Asn Ala Phe                610                                     620 CGT ACC AGCCAC TAC CCC TAT GCA GAG GAA GTG ATG CAG ATG TGT 2396 Arg Thr Ser His TyrPro Tyr Ala Glu Glu Val Met Gln Met Cys                                    630 GAC CGC TAT GGG ATT GTG GTC ATCGAT GAG TGT CCC GGC GTG GGG 2441 Asp Arg Tyr Gly Ile Val Val Ile Asp GluCys Pro Gly Val Gly                640                                     650 CTG GCG CTGCCG CAG TTC TTC AAC AAC GTT TCT CTG CAT CAC CAC 2486 Leu Ala Leu Pro GlnPhe Phe Asn Asn Val Ser Leu His His His                                    660 ATG CAG GTG ATG GAA GAA GTG GTGCGT AGG GAC AAG AAC CAC CCC 2531 Met Gln Val Met Glu Glu Val Val Arg ArgAsp Lys Asn His Pro                670                                     680 GCG GTC GTGATG TGG TCT GTG GCC AAC GAG CCT GCG TCC CAC CTA 2576 Ala Val Val Met TrpSer Val Ala Asn Glu Pro Ala Ser His Leu                                    690 GAA TCT GCT GGC TAC TAC TTG AAGATG GTG ATC GCT CAC ACC AAA 2621 Glu Ser Ala Gly Tyr Tyr Leu Lys Met ValIle Ala His Thr Lys                700                                     710 TCC TTG GACCCC TCG CGG CCT GTG ACC TTT GTG AGC AAC TCT AAC 2666 Ser Leu Asp Pro SerArg Pro Val Thr Phe Val Ser Asn Ser Asn                                    720 TAT GCA GCA GAC AAG GGG GCT CCGTAT GTG GAT GTG ATC TGT TTG 2711 Tyr Ala Ala Asp Lys Gly Ala Pro Tyr ValAsp Val Ile Gys Leu                730                                     740 AAC AGC TACTAC TCT TGG TAT CAC GAG TAC GGG CAC CTG GAG TTG 2756 Asn Ser Tyr Tyr SerTrp Tyr His Asp Tyr Gly His Leu Glu Leu                                    750 ATT CAG CTG CAG CTG GCC ACC CAGTTT GAG AAC TGG TAT AAG AAG 2801 Ile Gln Leu Gln Leu Ala Thr Gln Phe GluAsn Trp Tyr Lys Lys                760                                     770 TAT CAG AAGCCC ATT ATT CAG AGC GAG TAT GGA GCA GAA ACG ATT 2346 Tyr Gln Lys Pro IleIle Gln Ser Glu Tyr Gly Ala Glu Thr Ile                                    780 GCA GGG TTT CAC CAG GAT CCA CCTCTG ATG TTC ACT GAA GAG TAG 2891 Ala Gly Phe His Gln Asp Pro Pro Leu MetPhe Thr Glu Glu Tyr                790                                     800 CAG AAA AGTCTG CTA GAG CAG TAC CAT CTG GGT CTG GAT CAA AAA 2936 Gln Lys Ser Leu LeuGlu Gln Tyr His Leu Gly Leu Asp Gln Lys                                    810 CGC AGA AAA TAT GTG GTT GGA GAGCTC ATT TGG AAT TTT GCC GAT 2981 Arg Arg Lys Tyr Val Val Gly Glu Leu IleTrp Asn Phe Ala Asp                820                                     830 TTG ATG ACTGAA CAG TCA CCG ACG AGA GTG CTG GGG ATT AAA AAG 3026 Phe Met Thr Glu GlnSer Pro Thr Arg Val Leu Gly Asn Lys Lys                                    840 GGG ATG TTC ACT CGG CAG AGA CAACCA AAA AGT GCA GCG TTC CTT 3071 Gly Ile Phe Thr Arg Gln Arg Gln Pro LysSer Ala Ala Phe Leu                850                                     860 TTG CGA GAGAGA TAC TGG AAG ATT GCC AAT GAA ACC AGG TAT CCC 3116 Leu Arg Glu Arg TyrTrp Lys Ile Ala Asn Glu Thr Arg Tyr Pro                                    870 GAG TCA GTA GCC AAG TCA CAA TGTTTG GAA AAC AGC CCG TTT ACT 3161 His Ser Val Ala Lys Ser Gln Cys Leu GluAsn Ser Pro Phe Thr                880                                     890 TGAGCAAGACTGA TACCACCTGC GTGTCCCTTC CTCCCGCAGT CAGGGCGAGT 3214 . . .TCCACAGCAG CAGAACAAGT GCCTCCTGGA CTGTTCACGG CAGACCAGAA 3264 CGTTTCTGGCCTGGGTTTTG TGGTCATCTA TTCTAGCAGG GAACACTAAA 3314


17. A vector containing a nucleic acid as claimed in claim
 14. 18. Ahost cell containing a nucleic acid as claimed in claim 14 or a vectoras claimed in claim
 17. 19. A host cell as claimed in claim 18, which isa BHK, CHO, COS, HeLa, insect, tobacco plant, yeast or E.coli cell. 20.A transgenic mammal with the exception of a human, containing a DNA asclaimed in claim 14 or a vector as claimed in claim
 17. 21. A processfor preparing a compound as claimed in claim 1, which comprises a)introducing a nucleic acid as claimed in claim 14 or a vector as claimedin claim 17 into a host cell, b) cultivating the host cell, and c)isolating the compound.
 22. A process for preparing a compound asclaimed in claim 1, which comprises a) cultivating a host cell asclaimed in claim 18, and b) isolating the compound.
 23. The use of thecompound as claimed in claim 1 for the preparation of a pharmaceuticalor of a diagnostic aid.
 24. The use of the compound as claimed in claim1 for the preparation of a pharmaceutical for the treatment of cancer.25. A pharmaceutical containing a compound as claimed in claim
 1. 26. Adiagnostic aid containing a compound as claimed in claim 1.